应用聚合酶链反应从坦桑尼亚西北部成年人群的血清和干血斑卡样本中检测曼氏血吸虫 DNA。
Detection of Schistosoma mansoni DNA using polymerase chain reaction from serum and dried blood spot card samples of an adult population in North-western Tanzania.
机构信息
Medical Mission Institute, Hermann-Schell-Str. 7, 97074, Wuerzburg, Germany.
Department of Medical Parasitology and Entomology, School of Medicine, Catholic University of Health and Allied Sciences, P.O. Box 1464, Mwanza, Tanzania.
出版信息
Infect Dis Poverty. 2021 Feb 23;10(1):15. doi: 10.1186/s40249-021-00798-4.
BACKGROUND
Real-time polymerase chain reaction (PCR) is a sensitive and specific method for diagnosing schistosomiasis. However, this method should be performed in a laboratory, usually located distant from the sample collection site. Therefore, it is important to have fast sampling preservation methods, which allow simple transport prior to DNA extraction and amplification. The aim of this study was to verify if blood samples applied to filter paper are suitable for analysis of Schistosoma mansoni DNA by real-time PCR.
METHODS
A cross-sectional study was conducted among 100 study participants aged 17 to 70 years in a fishing village on the southern shore of Lake Victoria, Tanzania. Serum samples and ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood for preparation of dried blood spots (DBS) were collected to test for Schistosoma mansoni infection by real-time PCR. A combined diagnostic reference of positive results of serum-based real-time PCR and the Kato-Katz (KK) method was used for analysis. Sensitivity and negative predictive value (NPV) were calculated. The Wilcoxon signed-rank test was chosen to compare the mean cycle threshold (Ct) values from serum and DBS.
RESULTS
According to the reference, 92.5% S. mansoni positive samples were determined. The serum-based real-time PCR performed excellently with 95.4% sensitivity, whereas the DBS-based real-time PCR showed a low sensitivity (45.4%). The Ct-values were significantly higher in DBS (median: 37.3) than in serum samples (median: 27.5, P < 0.001), reflecting a lower parasite-specific DNA load on the filter cards. With increasing egg counts, an increase in sensitivity was observed for all methods. The POC-CCA test and the serum-based real-time PCR showed a sensitivity of 100% for medium and severe infections. The DBS real-time PCR showed a sensitivity of only 85.7% even for severe infections.
CONCLUSIONS
DBS-based real-time PCR did not provide good results in our study and therefore should not be recommended or must be tested concerning temperature of storage, storage duration, use of different filter papers and extraction methods before it is used in future studies. In contrast, our results showed that the POC-CCA test is a sensitive and precise test for detecting S. mansoni infections .
背景
实时聚合酶链反应（PCR）是一种用于诊断血吸虫病的敏感和特异的方法。然而，这种方法需要在实验室中进行，通常位于远离样本采集地点的地方。因此，拥有快速的采样保存方法非常重要，这使得在提取和扩增 DNA 之前可以进行简单的运输。本研究的目的是验证滤纸应用的血液样本是否适合实时 PCR 分析曼氏血吸虫 DNA。
方法
在坦桑尼亚维多利亚湖南岸的一个渔村，对 100 名 17 至 70 岁的研究参与者进行了一项横断面研究。采集血清样本和乙二胺四乙酸（EDTA）抗凝全血，用于制备干血斑（DBS），通过实时 PCR 检测曼氏血吸虫感染。采用基于血清的实时 PCR 和加藤法（KK）阳性结果的综合诊断参考标准进行分析。计算了敏感性和阴性预测值（NPV）。选择 Wilcoxon 符号秩检验来比较血清和 DBS 的平均循环阈值（Ct）值。
结果
根据参考标准，92.5%的曼氏血吸虫阳性样本被确定。基于血清的实时 PCR 具有出色的性能，敏感性为 95.4%，而基于 DBS 的实时 PCR 显示出较低的敏感性（45.4%）。DBS（中位数：37.3）中的 Ct 值明显高于血清样本（中位数：27.5，P<0.001），反映了滤纸上寄生虫特异性 DNA 载量较低。随着卵数的增加，所有方法的敏感性均有所提高。POC-CCA 检测和基于血清的实时 PCR 对中度和重度感染的敏感性均为 100%。即使对于严重感染，DBS 实时 PCR 的敏感性也仅为 85.7%。
结论
在我们的研究中，基于 DBS 的实时 PCR 未提供良好的结果，因此不应推荐或必须在未来的研究中测试其存储温度、存储持续时间、使用不同的滤纸和提取方法，然后再使用。相比之下，我们的结果表明，POC-CCA 检测是一种敏感而精确的检测曼氏血吸虫感染的方法。
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